Protein phosphorylation is an important mechanism for enzyme regulation and the transduction of extracellular signals across the cell membrane in eukaryotic cells. A wide variety of cellular substrates, including enzymes, membrane receptors, ion channels and transcription factors, can be phosphorylated in response to extracellular signals that interact with cells. A key enzyme in the phosphorylation of cellular proteins in response to hormones and neurotransmitters is cyclic AMP (cAMP)-dependent protein kinase (PKA). Upon activation by cAMP, PKA thus mediates a variety of cellular responses to such extracellular signals.
An array of PKA isozymes are expressed in mammalian cells. The PKA holoenzymes usually exist as inactive tetramers containing a regulatory (R) subunit dimer and two catalytic (C) subunits. Genes encoding three C subunits (Cα, Cβ and Cγ) and four R subunits (RIα, RIβ, RIIα and RIIβ) have been identified (see Takio et al. (1982) Proc. Natl. Acad. Sci. USA, 79:2544-2548; Lee et al. (1983) Proc. Natl. Acad. Sci. USA, 80:3608-3612; Jahnsen et al. (1996) J. Biol. Chem., 261:12352-12361; Clegg et al. (1988) Proc. Natl. Acad. Sci. USA, 85:3703-3707; and Scott (1991) Pharmacol. Ther., 50:123-145). The type I (RI) α and type II (RII) α subunits are distributed ubiquitously, whereas RIβ and RIIβ are present mainly in brain (see. e.g., Miki and Eddy (1999) J. Biol. Chem., 274:29057-29062). The type I PKA holoenzyme (RIα and RIβ) is predominantly cytoplasmic, whereas the majority of type II PKA (RIIα and RIIβ) associates with cellular structures and organelles (Scott (1991) Pharmacol. Ther., 50:123-145). Many hormones and other signals act through receptors to generate cAMP which binds to the R subunits of PKA and releases and activates the C subunits to phosphorylate proteins.
Because protein kinases and their substrates are widely distributed throughout cells, there are mechanisms in place in cells to localize protein kinase-mediated responses to different signals. One such mechanism involves subcellular targeting of PKAs through association with anchoring proteins, referred to as A-kinase anchoring proteins (AKAPs), that place PKAs in close proximity to specific organelles or cytoskeletal components and particular substrates, thereby providing for more specific PKA interactions and localized responses (see, e.g., Scott et al. (1990) J. Biol. Chem., 265:21561-21566; Bregman et al. (1991) J. Biol. Chem., 266:7207-7213; and Miki and Eddy (1999) J. Biol. Chem., 274:29057-29062). Anchoring not only places the kinase close to preferred substrates, but also positions the PKA holoenzyme at sites where it can optimally respond to fluctuations in the second messenger cAMP (Mochly-Rosen (1995) Science, 268:247-251; Faux and Scott (1996) Trends Biochem. Sci., 21:312-315; Hubbard and Cohen (1993) Trends Biochem. Sci., 18:172-177).
Up to 75% of type II PKA is localized to various intracellular sites through association of the regulatory subunit (RII) with AKAPs (see, e.g., Hausken et al. (1996) J. Biol. Chem., 271:29016-29022). RII subunits of PKA bind to AKAPs with nanomolar affinity (Carr et al. (1992) J. Biol. Chem., 267:13376-13382), and many AKAP-RII complexes have been isolated from cell extracts. RI subunits of PKA bind to AKAPs with only micromolar affinity (Burton et al. (1997) Proc. Natl. Acad. Sci. USA 94:11067-11072). Evidence of binding of a PKA RI subunit to an AKAP has been reported (Miki and Eddy (1998) J. Biol. Chem., 273:34384-34390) in which RIα-specific and RIα/RIIα dual specificity PKA anchoring domains were identified on FSC1/AKAP82. Additional dual specific AKAPs, referred to as D-AKAP1 and D-AKAP2, which interact with the type I and type II regulatory subunits of PKA have also been reported (Huang et al. (1997) J. Biol. Chem., 272:8057-8064; Huang et al. (1997) Proc. Natl. Acad. Sci. USA, 94:11184-11189).
More than 20 AKAPs have been reported in different tissues and species. Complementary DNAs (cDNAs) encoding AKAPs have been isolated from diverse species, ranging from Caenorhabditis elegans and Drosophilia to human (see, e.g., Colledge and Scott (1999) Trends Cell Biol., 9:216-221). Regions within AKAPs that mediate association with RII subunits of PKA have been identified. These regions of approximately 10-18 amino acid residues vary substantially in primary sequence, but secondary structure predictions indicate that they are likely to form an amphipathic helix with hydrophobic residues aligned along one face of the helix and charged residues along the other (Carr et al. (1991) J. Biol. Chem., 266:14188-14192; Carr et al. (1992) J. Biol. Chem., 267:13376-13382). Hydrophobic amino acids with a long aliphatic side chain, e.g., valine, leucine or isoleucine, may participate in binding to RII subunits (Glantz et al. (1993) J. Biol. Chem., 268:12796-12804).
Many AKAPs also have the ability to bind to multiple proteins, including other signalling enzymes. For example, AKAP79 binds to PKA, protein kinase C (PKC) and the protein phosphatase calcineurin (PP2B) (Coghlan et al. (1995) Science, 267:108-112 and Klauck et al. (1996) Science, 271:1589-1592). Therefore, the targeting of AKAP79 to neuronal postsynaptic membranes brings together enzymes with opposite catalytic activities in a single complex.
AKAPs thus serve as potential regulatory mechanisms that increase the selectivity and intensity of a cAMP-mediated response. There is a need, therefore, to identify and elucidate the structural and functional properties of AKAPs in order to gain a complete understanding of the important role these proteins play in the basic functioning of cells.